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CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

Wilson JL, Wareham LK, McLean S, Begg R, Greaves S, Mann BE, Sanguinetti G, Poole RK - Antioxid. Redox Signal. (2015)

Bottom Line: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy.A full understanding of the actions of CORMs is vital to understand their toxic effects.This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom .

ABSTRACT

Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools.

Results: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3.

Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects.

Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

No MeSH data available.


Related in: MedlinePlus

Heme-deficientEscherichia coliare more CORM-sensitive than isogenic wild-type or heme-reconstituted bacteria. Cultures were grown anaerobically and stressed with 100 μM (open circles), 200 μM (closed triangles), and for growth studies only, 300 μM (open triangles) CORM-3 at an OD600 of ∼0.1 (dashed line). Growth and viability of CORM-3-treated cultures were compared with control cultures (nothing added, closed circles). To determine the effects of CORM-3 on growth, hourly OD600 readings were taken for 6 h post-addition of the compound, followed by a final 24 h reading. Wild type (A), heme-deficient mutant (B), and cells reconstituted for heme (C) by adding δ-ALA (0.1 mM final concentration). For viability assays, a sample was taken immediately before addition of the compound, followed by sampling every hour for 4 h post-stress and at 24 h to complete the experiment. Wild type (D), heme-deficient mutant (E), and cells reconstituted for heme (F) by adding δ-ALA. Data show patterns seen in ≥3 biological replicates. Viability data are plotted as means±SEM from ≥3 individual spots. Note that the scale on the y-axis is logarithmic in base 10, hence 1e+3=1000.
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f1: Heme-deficientEscherichia coliare more CORM-sensitive than isogenic wild-type or heme-reconstituted bacteria. Cultures were grown anaerobically and stressed with 100 μM (open circles), 200 μM (closed triangles), and for growth studies only, 300 μM (open triangles) CORM-3 at an OD600 of ∼0.1 (dashed line). Growth and viability of CORM-3-treated cultures were compared with control cultures (nothing added, closed circles). To determine the effects of CORM-3 on growth, hourly OD600 readings were taken for 6 h post-addition of the compound, followed by a final 24 h reading. Wild type (A), heme-deficient mutant (B), and cells reconstituted for heme (C) by adding δ-ALA (0.1 mM final concentration). For viability assays, a sample was taken immediately before addition of the compound, followed by sampling every hour for 4 h post-stress and at 24 h to complete the experiment. Wild type (D), heme-deficient mutant (E), and cells reconstituted for heme (F) by adding δ-ALA. Data show patterns seen in ≥3 biological replicates. Viability data are plotted as means±SEM from ≥3 individual spots. Note that the scale on the y-axis is logarithmic in base 10, hence 1e+3=1000.

Mentions: We first verified that the hemA strain constructed by P1 transduction lacked cytochromes (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/ars) and was unable to grow on nonfermentable substrates, such as glycerol or succinate (not shown). Cultures of the heme-deficient mutant and wild type strains were then stressed with CORM-3 or inactive CORM-3 (iCORM-3). Micromolar concentrations of CORM-3 resulted in a concentration-dependent slowing of growth (Fig. 1A–C) for the wild-type strain, hemA mutant, and the mutant after reconstitution with δ-ALA. Wild-type cultures stressed with 100 μM CORM-3 showed a marginally increased doubling time (0.81±0.18 h) compared with the control (0.79±0.13 h) but, at 200 and 300 μM CORM-3, growth was prevented for 5 h. At these concentrations, cultures showed some recovery between 8–24 h, but cell densities did not reach the level of control or 100 μM-treated cultures (Fig. 1A). Similar results were obtained for the hemA mutant; 100 μM CORM-3 increased the doubling time from 2.0±0.33 h (control) to 3.6±1.3 h. Unlike the control, mutant cultures did not recover even after 28 h of incubation with the CORM (Fig. 1B).


CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli.

Wilson JL, Wareham LK, McLean S, Begg R, Greaves S, Mann BE, Sanguinetti G, Poole RK - Antioxid. Redox Signal. (2015)

Heme-deficientEscherichia coliare more CORM-sensitive than isogenic wild-type or heme-reconstituted bacteria. Cultures were grown anaerobically and stressed with 100 μM (open circles), 200 μM (closed triangles), and for growth studies only, 300 μM (open triangles) CORM-3 at an OD600 of ∼0.1 (dashed line). Growth and viability of CORM-3-treated cultures were compared with control cultures (nothing added, closed circles). To determine the effects of CORM-3 on growth, hourly OD600 readings were taken for 6 h post-addition of the compound, followed by a final 24 h reading. Wild type (A), heme-deficient mutant (B), and cells reconstituted for heme (C) by adding δ-ALA (0.1 mM final concentration). For viability assays, a sample was taken immediately before addition of the compound, followed by sampling every hour for 4 h post-stress and at 24 h to complete the experiment. Wild type (D), heme-deficient mutant (E), and cells reconstituted for heme (F) by adding δ-ALA. Data show patterns seen in ≥3 biological replicates. Viability data are plotted as means±SEM from ≥3 individual spots. Note that the scale on the y-axis is logarithmic in base 10, hence 1e+3=1000.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492677&req=5

f1: Heme-deficientEscherichia coliare more CORM-sensitive than isogenic wild-type or heme-reconstituted bacteria. Cultures were grown anaerobically and stressed with 100 μM (open circles), 200 μM (closed triangles), and for growth studies only, 300 μM (open triangles) CORM-3 at an OD600 of ∼0.1 (dashed line). Growth and viability of CORM-3-treated cultures were compared with control cultures (nothing added, closed circles). To determine the effects of CORM-3 on growth, hourly OD600 readings were taken for 6 h post-addition of the compound, followed by a final 24 h reading. Wild type (A), heme-deficient mutant (B), and cells reconstituted for heme (C) by adding δ-ALA (0.1 mM final concentration). For viability assays, a sample was taken immediately before addition of the compound, followed by sampling every hour for 4 h post-stress and at 24 h to complete the experiment. Wild type (D), heme-deficient mutant (E), and cells reconstituted for heme (F) by adding δ-ALA. Data show patterns seen in ≥3 biological replicates. Viability data are plotted as means±SEM from ≥3 individual spots. Note that the scale on the y-axis is logarithmic in base 10, hence 1e+3=1000.
Mentions: We first verified that the hemA strain constructed by P1 transduction lacked cytochromes (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/ars) and was unable to grow on nonfermentable substrates, such as glycerol or succinate (not shown). Cultures of the heme-deficient mutant and wild type strains were then stressed with CORM-3 or inactive CORM-3 (iCORM-3). Micromolar concentrations of CORM-3 resulted in a concentration-dependent slowing of growth (Fig. 1A–C) for the wild-type strain, hemA mutant, and the mutant after reconstitution with δ-ALA. Wild-type cultures stressed with 100 μM CORM-3 showed a marginally increased doubling time (0.81±0.18 h) compared with the control (0.79±0.13 h) but, at 200 and 300 μM CORM-3, growth was prevented for 5 h. At these concentrations, cultures showed some recovery between 8–24 h, but cell densities did not reach the level of control or 100 μM-treated cultures (Fig. 1A). Similar results were obtained for the hemA mutant; 100 μM CORM-3 increased the doubling time from 2.0±0.33 h (control) to 3.6±1.3 h. Unlike the control, mutant cultures did not recover even after 28 h of incubation with the CORM (Fig. 1B).

Bottom Line: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy.A full understanding of the actions of CORMs is vital to understand their toxic effects.This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

View Article: PubMed Central - PubMed

Affiliation: 1 Department of Molecular Biology and Biotechnology, The University of Sheffield , Sheffield, United Kingdom .

ABSTRACT

Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools.

Results: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3.

Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects.

Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy.

No MeSH data available.


Related in: MedlinePlus